METTL3-mediated m6A modification of OTUD1 aggravates press overload induced myocardial hypertrophy by deubiquitinating PGAM5

Background: Pathological cardiac hypertrophy, a condition that contributes to heart failure, is characterized by its intricate pathogenesis. The meticulous regulation of protein function, localization, and degradation is a crucial role played by deubiquitinating enzymes in cardiac pathophysiology. This study clarifies the participation and molecular mechanism of OTUD1 (OTU Deubiquitinase 1) in pathological cardiac hypertrophy. Methods: We generated a cardiac-specific Otud1 knockout mouse line (Otud1-CKO) and adeno-associated virus serotype 9-Otud1 mice to determine the role of Otud1 in cardiac hypertrophy. Its impact on cardiomyocytes enlargement was investigated using the adenovirus. RNA immunoprecipitation was used to validate the specific m6a methyltransferase interacted with OTUD1 transcript. RNA sequencing in conjunction with immunoprecipitation-mass spectrometry analysis was employed to identify the direct targets of OTUD1. A series of depletion mutant plasmids were constructed to detect the interaction domain of OTUD1 and its targets. Results: Ang II-stimulated neonatal rat cardiac myocytes and mice hearts subjected to transverse aortic constriction (TAC) showed increased protein levels of Otud1. Cardiac hypertrophy and dysfunction were less frequent in Otud1-CKO mice during TAC treatment, while Otud1 overexpression worsened cardiac hypertrophy and remodeling. METTL3 mediated m6A modification of OTUD1 transcript promoted mRNA stability and elevated protein expression. In terms of pathogenesis, Otud1 plays a crucial role in cardiac hypertrophy by targeting Pgam5, leading to the robust activation of the Ask1-p38/JNK signal pathway to accelerate cardiac hypertrophy. Significantly, the pro-hypertrophy effects of Otud1 overexpression were largely eliminated when Ask1 knockdown. Conclusion: Our findings confirm that targeting the OTUD1-PGAM5 axis holds significant potential as a therapeutic approach for heart failure associated with pathological hypertrophy.

The data collection and analysis were conducted by two observers who were blinded to the group assignment and treatment of the animals.Male mice were exclusively chosen for all animal experiments in this study.This decision was based on previous research indicating that estrogen and low testosterone levels might confer protection against cardiac fibrosis.By utilizing male mice, we aimed to induce significant tissue failure and cardiac fibrosis through the challenge of transverse aortic constriction (TAC), thereby ensuring robust results.

Cardiac hypertrophy model
Pathological cardiac hypertrophy is conducted by TAC surgery.Mice (8-10 weeks) were anesthetized.The neck skin was cut to expose the trachea and the second rib was cut along the sternum.After the separation of thymus, the transverse aortic arch was exposed, and was ligated with 6-0 silk against a 27G needle.The chest was closed after the removal of needle.Mice in the sham group underwent similar operation without ligation of transverse aortic arch.Four weeks after TAC, heart function was measured by echocardiography as previously described.After euthanasia, the heart, lung and tibia of mice were dissected and measured, and the ratios of heart weight (HW)/body weight (BW) (mg/g), HW/tibia length (TL) (mg/mm) and lung weight (LW)/body weight (mg/g) were calculated.All operations and subsequent analyses were performed in a blinded fashion.

AAV9-Otud1 overexpression mice
The OTUD1 and GFP genes were amplified from cDNA and ligated into the vector pAAV-cTnT-WPRE (OBiO Technology, Shanghai, China), and constructed plasmid was packaged into AAV9 vectors carrying the cardiac troponin T (cTnT) promoter to drive the expression of GFP (pAAV-cTnT-GFP-WPRE) and Otud1 (pAAV-cTnT-GFP-P2A-Otud1-3xFLAG-WPRE).Viral solution (7.5 × 10^11 vg/ml, 200 μl/mouse) was injected via the tail vein after the mice were anaesthetized.Two weeks after the AAV9 injection, the animals were randomly assigned to either a sham operation group or a TAC group for another four weeks.Surgeries and subsequent analyses were performed in a blinded fashion.

Administration of ASK1 inhibitor GS-444217
TAC mice were randomized to be administered vehicle only or GS-444217 (0.2% in chow) for 4 weeks.

Administration of METTL3 inhibitor STM2457
TAC mice were randomized to be administered vehicle only or STM2457 (50 mg/kg) daily for 4 weeks.

Administration of PGAM5 inhibitor LFHP-1c
TAC mice were randomized to be administered vehicle only or STM2457 (3 mg/kg) daily for 4 weeks.

Hematoxylin-Eosin staining
Paraffin-embedded sections of heart were dewaxed, dehydrated, rinsed in distilled water.Then, the sections were stained with hematoxylin solution for 3 min, washed with running tap water for 5 min and counterstained in eosin alcoholic solution for 1 min.At last, the sections were dehydrated in increasing concentrations of ethyl alcohol and cleared in xylene for 2 min.A microscope was used to taken pictures which were evaluated by two investigators blinded to the grouping.

Masson's trichrome staining
Masson's Trichrome Stain Kit (G1340, Solarbio, Beijing, China) was used to perform Masson's trichrome staining.Briefly, after deparaffinization and rehydration, the sections were stained in a Weigert iron hematoxylin solution for 5 min, differentiated with acidic ethanol solution for 10 s and treated with Masson bluing solution for 3 min.Then, the sections were incubated with Ponceau S solution for 5 min and rinsed with 1% acetic acid for 1 min.Next, they were placed in 2.5% phosphotungstic acid solution for 10 min and rinsed with 1% acetic acid.Finally, the tissue sections were immersed in aniline blue solution, dehydrated in ethanol, cleared in xylene and sealed with resinene.

Picrosirius red staining
Heart sections were dewaxed in xylene for 15 min (twice), dehydrated in gradient ethanol and washed with running tap water for 30s.Then, the sections were immersed in 0.1 % picrosirius red staining for 90 min and washed in running tap water for 10s.After dyeing, sections were treated with 0.5% acetic acid for 20 s, dehydrated in ethanol and cleared in xylene.The sections were sealed with resinene before light microscope observation.

Histological analysis
Image-Pro Plus (Version 6.0) was used to obtain images from sections stained with hematoxylineosin, Masson and Picrosirius red, and the cross-sectional areas of cardiomyocytes and fibrotic areas were evaluated.

Primary cardiomyocytes isolation and cell treatment
Primary neonatal rat cardiac myocytes (NRCMs) were isolated from hearts of Sprague-Dawley rats within 3 days.Briefly, hearts were minced, digested in 0.2 % Collagenase II (LS0004176, Worthington Bio, USA) for 15 min.The supernatant was obtained and the sediment was redigested for several times until disappeared.After removing the fibroblasts, NRCMs were cultured in High Glucose DMEM (11965092, Gibco) containing 10 % fetal bovine serum (A5669801, Gibco) and 1 % penicillin/streptomycin (C0222, Beyotime Biotechnology, Shanghai, China).NRCMs were infected with specified adenoviruses at a multiplicity of infection of 50 for 24 hours, followed by stimulated with 10μM Ang Ⅱ (HY-13948, MedChemExpress, USA) for 48 hours.

Immunofluorescence staining
NRCMs were fixed with 4 % paraformaldehyde at room temperature for 20 min, washed with PBS for 3 min (three times), incubated with 0.2% Triton-X 100 (93443, Merck KGaA, Darmstadt, Germany) for 15 min at room temperature, blocked in blocking buffer (P0260, Beyotime Biotechnology, Shanghai, China) for 15 min.Then, NRCMs were incubates with primary antibody at 4℃ overnight and corresponding secondary antibody for 1h at room temperature.DAPI ( C1005, Beyotime Biotechnology, Shanghai, China) was used to stain cell nucleus .Images were obtained via fluorescence microscope.

Western blot analysis
The RIPA buffer (P0013C, Beyotime Biotechnology, Shanghai, China) was used to extract protein from heart tissues and myocardial cells.BCA Protein Assay Kit (P0012, Beyotime Biotechnology, Shanghai, China) was used to detect protein concentration.After that, equal amount of protein was added and separated by 10% SDS-PAGE (PG112, Epizyme Biotech, Shanghai, China).Next, protein was transferred to PVDF membrane (88520, Thermo Fisher Scientific Inc, Carlsbad, CA, USA).Then, the membrane was blocked with 5% nonfat milk for one hour and incubated with primary antibody overnight at 4℃.After that, the membrane was rinsed with TBST solution for 15 min (three times) and incubated with HRP-conjugated secondary antibody at room temperature for one hour.Finally, the membrane was immersed with the chemiluminescence (ECL) reagent and the relative expression of protein was detected by an ECL system.The primary antibodies used was showed in Supplementary Table S1.

Quantitative real-time PCR (qPCR)
TRIzol reagent (15596026, Thermo Fisher Scientific Inc, Carlsbad, CA, USA) was used to isolated total RNA from NRCMs and heart tissue according to manufacturer's instructions.The first-strand cDNA was synthesized from 2 μg of total RNA by PrimeScript RT Master Mix (RR036A, TaKaRa, Japan).The mRNA expression levels of related genes were analyzed on a LightCycler ® 480Ⅱ system (Roche, Basel, Switzerland) using TB Green (RR820Q, TaKaRa, Japan).The levels of detected mRNA were calculated by 2 -ΔΔ Ct method and normalized to the amount of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH).The sequences of the qPCR primers were listed in Supplementary Table S2.

N6-methyladenosine (m6A) dot blot
The isolated total RNA was heated at 95°C for 3 min to destruct the secondary structure.Then, the RNA samples were transferred to Amersham Hybond-N + membranes (YA1760, Solarbio Life Science, Beijing, China) followed by ultraviolet cross-linking for 60 min.Then, the membranes were blocked in 5% nonfat milk (dissolved in TBST) for 60 min and incubated with m6A antibody (A19841, ABclonal Technology, Wuhan, China) at 4°C overnight.Next, the membranes were incubated with HRP-conjugated secondary antibody and visualized by chemiluminescence method.

RNA immunoprecipitation coupled with quantitative reverse transcription polymerase chain reaction (RIP-qPCR)
RIP-qRT-PCR was performed using Magna RIP™ Kit (17-704, Merck KGaA, Darmstadt, Germany) according to manufacturer's instruction.Briefly, after specified treatment, cells (2 × 10^7 cells per RIP reaction) were harvested and lysed in a buffer containing RNase and protease inhibitor.Then, cell lysis was treated with DNase and added with indicated antibody and corresponding IgG for overnight at 4°C overnight.The protein/RNA complexes were hooked by magnetic protein A/G beads.Finally, the coprecipitated RNA was isolated and reverse transcribed using the protocol described above followed by qRT-PCR.The primers used for m6A RIP-qPCR analysis was listed in Supplementary Table S3.

m6A RNA Methylation Quantification
The overall methylation m6A content was detected using the m6A RNA Methylation Assay Kit (Abcam, MA, USA), following the manufacturer's instructions.In brief, 200 ng of total RNA was added to each reaction, along with the diluted capture antibody, detection antibody solution, and enhancer solution.The m6A level was quantified using colorimetry, with the absorbance of each reaction measured at 450 nm.

RNA stability
NRCMs were infected with targeted or control adenovirus and subjected to actinomycin D treatment (5 μg/mL, MCE).At indicated time points, cells were collected and total RNA was extracted using the miRNeasy Kit (217084, Qiagen).The half-life (t 1/2 ) of mRNA was analyzed and calculate by qRT-PCR as described above.

Coimmunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)
To investigate the underling mechanisms of OTUD1 induced pro-hypertrophic effects, Co-IP was carried out using commercially available kit (abs955, Absin Biotechnology, Shanghai, China).
NRCMs or 293 cells were washed with PBS.Heart tissues were treated with liquid nitrogen and ground into powder.The lysis buffer with 1 mM PMSF (abs9146, Absin Biotechnology, Shanghai, China) was added to cells or tissue powder at 4℃ for 20min.The lysate were subjected to ultrasonic crushing (FB50220, Thermo Fisher) for 5s (3 times, 10s interval, 40% max power) on the ice and centrifuged (14000 g, 4℃, 10 min) to obtain supernatant.The protein concentration was detected by using BCA assay kit (P0012, Beyotime Biotechnology, Shanghai, China).For removing the nonspecific binding proteins, a total of 500μl (1 ug/μl) protein was added with 5 μl Protein A and Protein G agarose beads, and incubated at 4℃ for 60 min on a rotating machine (20 rpm).The newly acquired supernatant were transferred to new centrifugal tubes.For IP group, 1 μg targeted monoclonal antibody was used.For Input group, 1 μg IgG was used.After antigen-antibody combination (4℃, 12h, 20rpm), 5 μl Protein A and Protein G agarose beads were added to link antibody-proteins complex for 2 h at 4 °C with rotation at 20 rpm.After that, the supernatant and beads-antibody-proteins complex were separated by centrifugation (4℃, 1 min, 12000g) followed by three times washing steps (4℃, 1 min, 12000g).The sediments were resuspended with 20 μl SDS buffer, boiled at 100℃ for 5 min and centrifugated for 1 min at 4℃at 12000g.Finally, the supernatants were SDS-PAGE analysis and immunoblotting described above.
For LC-MS/MS, the immunoprecipitation complex was separated by SDS-PAGE and then set to Wuhan Metware Biotechnology (Wuhan, China) for further analysis.

Ubiquitination assays
After pretreatment, cultured NRCMs, 293 cells or heart tissues was subjected to lysis and IP assays with indicated antibodies, followed by western blot.

Single cell RNA sequencing analysis
The scRNA datasets of the human heart (GSE161470, GSE145154, and GSE161153) were obtained from the publicly available GEO database (https://www.ncbi.nlm.nih.gov/geo/).Subsequently, four left ventricular control samples from GSE161470, three left ventricular HF samples from GSE145154, and one left ventricular sample with HF from GSE161153 were selected for further analysis.This analysis was conducted using R (version 4.3.1)and the Seurat package (version 4.1.1).
To form large gene expression matrices, the "merge" function was employed.Cells meeting the criteria of having more than 500 genes, less than 5,000 genes, and less than 20% mitochondrial genes were retained for subsequent analysis.Gene expression lists were normalized using the "NormalizeData" function and further scaled.Subsequently, the "vst" method was used for each sample to identify 2,000 highly variable genes.According to the highly variable genes, principal component analysis was applied to identify significant principal components (PCs), which were visualized using the ElbowPlot function.Since scRNA data were collected from three different research groups, the "Harmony" package (version 0.1.0)was used to correct the batch effect.We employed a sample of 20 PCs to conduct UMAP analysis.The classification of fifteen cell clusters was accomplished through the utilization of the "FindClusters" function, employing a resolution of 0.5.To identify DEGs within each cell cluster, we employed the "FindAllMarkers" function with a threshold of 0.25.The annotation of cell types was performed by considering the top five DEGs for each cell cluster, as well as referencing a previously published article 23 and utilizing the CellMarker database (http://bio-bigdata.hrbmu.edu.cn/CellMarker/).

RNA transcriptome sequencing
After infection of Ad-Otud1 and Ad-GFP, the total RNA was isolated from NRCMs (three independent samples per group) and sent to Wuhan Metware Biotechnology (Wuhan, China) for RNA sequencing, including RNA detection, library construction, and computer sequencing.After removing of reads with low quality, clean reads were mapped to the UCSC rn6 genomes using HISAT2 software (version 2.2.1) and transferred to the BAM format using samtools (version 1.6).
Then, the count matrix was created by featureCounts (version 2.0.3) and input into R (version 4.2.3).

Statistical analyses
Data were analyzed by GraphPad Prism 9 (GraphPad Software, Inc., CA, USA).All experiments were performed in triplicate unless stated otherwise.Values are presented as mean ± SD or median ± interquartile range.The Shapiro-Wilk test was used to evaluate normal distribution of data.For two group comparison, unpaired two-tailed Student's t-test (normal distribution) and Mann-Whitney test (skewed normal distribution) were used.For three or more groups comparisons, oneway ANOVA followed by Tukey post hoc analysis (normal distribution, homogeneity of variance), two-way ANOVA followed by Bonferroni post hoc test (normal distribution, homogeneity of variance, multi-factors), one-way ANOVA followed by Tamhane T2 post hoc analysis (normal distribution, heteroscedasticity) and Kruskal-Wallis test followed by Dunn post hoc analysis (skewed normal distribution) were used.The exponential decay equation models for mRNA halflife were developed using GraphPad Prism 9.The statistical tests used for the data in each figure are listed in the related legends.The sample size of mice in each experiment is indicated in related legends and determined based on previous studies 10,35 .No statistical method was used to predetermine the sample size.S1 S3 The primers used for m6A RIP-qPCR analysis.